人PSF蛋白的原核表达及分析鉴定
首发时间:2010-12-22
摘要:PSF(polypyrimidine tract binding protein-associated splicing factor, PSF)蛋白是一种多功能的DNA/RNA结合蛋白,它能够抑制原癌基因的表达,在人和小鼠中具有肿瘤抑制的作用。以人成纤维细胞总RNA为材料,利用PSF特异性引物通过RT-PCR的方法扩增得到PSF蛋白的编码序列,将其克隆到原核表达载体pET-28a(+)中构建重组表达质粒pET-28-PSF,转化大肠杆菌BL21(DE3)并诱导表达。SDS-PAGE及免疫印记分析表明PSF能够在大肠杆菌中以可溶形式表达。同时凝胶阻滞分析显示原核表达的PSF重组蛋白在体外条件下能够与mVL30-1 RNA结合,说明PSF的原核表达产物具有生物学活性,这为进一步研究PSF蛋白的生物学功能奠定了基础。
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Prokaryotic Expression, Analysis and Identification of Human Polypyrimidine Tract Binding Protein-associated Splicing Factor (PSF)
Abstract:Polypyrimidine tract binding protein-associated splicing factor (PSF), a multifunctional RNA/DNA-binding protein, can suppress the expression of proto-oncogene and function as tumor suppressor protein (TSP) in human and mouse. Using total RNA extracted from human fibroblasts as material, PSF-encoding cDNA was synthesized and inserted into vector pET-28a(+) to construct the recombinant plasmid pET-28-PSF. The PSF fused with His-tag was expressed in E.coli BL21 (DE3) by IPTG induction and then purified by affinity chromatography. The results of SDS-PAGE and Western blotting indicated the dissoluble His-tagged PSF could be produced with prokaryotic expression system. At last, the RNA-binding capacity of recombinant PSF to mVL30-1 RNA was determined by Gel-shift assay. Our results showed that recombinant PSF with biological activity can be produced with prokaryotic expression system, which will be the foundation of in-depth research on its function.
Keywords: PSF tumor prokaryotic expression
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